Modeling Tyrosinase Monooxygenase Activity. Spectroscopic and

Giuseppe Battaini, Enrico Monzani, Angelo Perotti, Cristina Para, Luigi Casella, Laura Santagostini, Michele Gullotti, Renée Dillinger, Christian Nä...
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Inorg. Chem. 1998, 37, 6597-6605

6597

Modeling Tyrosinase Monooxygenase Activity. Spectroscopic and Magnetic Investigations of Products Due to Reactions between Copper(I) Complexes of Xylyl-Based Dinucleating Ligands and Dioxygen: Aromatic Ring Hydroxylation and Irreversible Oxidation Products† Debalina Ghosh and Rabindranath Mukherjee*,‡ Department of Chemistry, Indian Institute of Technology, Kanpur 208 016, India ReceiVed October 29, 1997

A full account of a chemical system possessing features that mimic the reactivity aspects of tyrosinase is presented. Using dinucleating ligands with a m-xylyl spacer three new dicopper(I) complexes have been synthesized and their reactivity with dioxygen investigated. The six-membered chelate ring forming ligands provide only two nitrogen coordinations to each copper. The complexes [CuI2L(CH3CN)2]X2 (X ) ClO4- (1a), SbF6- (1b)) and [CuI2(L-NO2)(CH3CN)2][SbF6]2 (1c) [L ) R,R′-bis[N-methyl-N-(2-pyridylethyl)amino]-m-xylene; L-NO2 ) paranitro derivative of L] have been characterized by IR and 1H NMR spectroscopy. The reaction of O2 with 1a-c in CH2Cl2 or THF is instantaneous and causes stoichiometric xylyl hydroxylation reactions producing phenol products. Thus 1a produces phenoxo-/hydroxo-bridged product [CuII2(L-O)(OH)][ClO4]2 (2a). The existence of putative peroxo-dicopper(II) species could not be detected even at -80 °C. A trend is observed for the extent of aromatic ring hydroxylation (298 K): CH3CN ∼ DMF > CH3OH . CH2Cl2. Cyclic voltammetric experiment of 1a in DMF reveals an appreciably low redox potential (E1/2 ) -0.26 V vs SCE) for the CuII2/CuI2 redox process. Variable-temperature (25-300 K) magnetic susceptibility measurements establish that the copper(II) centers in 2a and the dihydroxo-bridged complex [CuII2L′(OH)2][ClO4]2 (2b) [formed due to an impurity (L′) present during the synthesis of L following Method A; L′ ) bis[R,R′-bis(N-methyl-N-(2-pyridylethyl)amino)-mxylene]methylamine] are antiferromagnetically coupled, with 2a considerably more coupled than 2b. Reaction of 1a with O2 in CH2Cl2 (298 K) produces an additional unhydroxylated product of composition [CuII2L(OH)(OH2)][ClO4]3‚2H2O‚0.5HCl (3a). In agreement with its proposed hydroxo-/aquo-bridged structure, 3a is weakly antiferromagnetically coupled. In CH3CN solution, 3a rearranges to generate a doubly hydroxo-bridged species [CuII2L(OH)2]2+. Using a solution-generated dicopper(I) complex of a closely similar ligand (L′′) providing fivemembered chelate ring, the reactivity toward dioxygen was also investigated. It produces only an irreversibly oxidized product of composition CuII2L′′(OH)(ClO4)3(H2O)2 (3b) (L′′ ) R,R′-bis[N-methyl-N-(2-pyridylmethyl)amino]-m-xylene). For 3b the copper(II) centers are almost uncoupled.

Introduction The activation of molecular oxygen by copper plays a central role in synthetically useful stoichiometric and catalytic oxidative conversions of organic molecules and in biological systems.1-6 Hemocyanin (Hc) and tyrosinase (Tyr) are dinuclear coppercontaining proteins that bind or activate dioxygen. Hc functions as a dioxygen carrier, and Tyr, a monooxygenase, which hydroxylates monophenols (tyrosine) and further oxidizes the o-diphenol to an o-quinone. The structures of both deoxy and † Dedicated to Professor Richard H. Holm on the occasion of his 65th birthday. ‡ E-mail: [email protected]. (1) (a) Kitajima, N.; Moro-oka, Y. Chem. ReV. 1994, 94, 737. (b) Karlin, K. D.; Gultneh, Y. Prog. Inorg. Chem. 1987, 35, 219. (2) (a) Karlin, K. D.; Kaderli, S.; Zuberbu¨hler, A. D. Acc. Chem. Res. 1997, 30, 139. (b) Tolman, W. B. Acc. Chem. Res. 1997, 30, 227. (3) (a) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. ReV. 1996, 96, 2563. (b) Solomon, E. I.; Lowery, M. D. Science 1993, 259, 1575. (c) Solomon, E. I.; Baldwin, M. J.; Lowery, M. D. Chem. ReV. 1992, 92, 521. (4) (a) Holm, R. H.; Kennepohl, P.; Solomon, E. I. Chem. ReV. 1996, 96, 2239. (b) Magnus, K. A.; Ton-That, H.; Carpenter, J. E. Chem. ReV. 1994, 94, 727. (5) (a) Karlin, K. D.; Tyekla´r, Z. AdV. Inorg. Biochem. 1994, 9, 123. (b) Tyekla´r, Z.; Karlin, K. D. Acc. Chem. Res. 1989, 22, 241. (6) (a) Kitajima, N. AdV. Inorg. Chem. 1992, 39, 1. (b) Sorrell, T. N. Tetrahedron 1989, 45, 3.

oxy forms of Hc have been determined by X-ray crystallography.3,4 In oxyHc the presence of a novel (µ-η2:η2peroxo)dicopper(II) species has been identified. This Cu2O2 unit provides a strong antiferromagnetic coupling in the binuclear site.3 Each copper center has a distinctly elongated histidine coordination (Cu-N bond length 2.26 Å) above and below the Cu-O2-Cu plane and has two equatorial histidine coordinations (Cu-N bond length 2.00 Å). No X-ray crystal structure has been available to date, but spectroscopic and EXAFS measurements suggest3 that the active site of Tyr is similar to that of Hc. The difference in biological function between the two proteins is that the protected buried active site in Hc is instead accessible to phenolic substrates in Tyr.2a,3 The elegant biomimetic studies from a number of laboratories1,2,5-8 have largely contributed to the understanding of the oxidized forms of Hc and Tyr. Considerable progress has been made in the chemical modeling of Tyr-like monooxygenase activity.1,5,6,9-18 Karlin et al. reported the first10 model consisting of a binucleating ligand having a m-xylyl spacer between the two tridentate bis[2(7) Sorrell, T. N.; Allen, W. E.; White, P. S. Inorg. Chem. 1995, 34, 952. (8) Lynch, W. E.; Kurtz, D. M., Jr.; Wang, S.; Scott, R. A. J. Am. Chem. Soc. 1994, 116, 11030. (9) Spodine, E.; Manzur, J. Coord. Chem. ReV. 1992, 119, 171.

10.1021/ic9713689 CCC: $15.00 © 1998 American Chemical Society Published on Web 12/01/1998

6598 Inorganic Chemistry, Vol. 37, No. 26, 1998 (2-pyridylethyl)amine] coordination units. Hydroxylation reactions have also been seen with other modified xylyl systems of Schiff bases.13-17 Interestingly, these latter systems provide only two N-coodinations per copper. Martell and co-workers18 demonstrated the first successful use of a xylyl-based macrocyclic ligand derived from diethylenetriamine to bring about aromatic ring hydroxylation. Following the observations concerning (i) the stereochemistry of copper-nitrogen bonding in the crystal structures of Hc, (ii) the literature reports on Tyr models of xylyl-based Schiff base systems, and (iii) the fact that the active site of Tyr is more accessible to substrates, we initiated a program to systematically investigate Tyr-like activity using new m-xylyl-based ligand systems of the open-chain type19a or of the macrocyclic type,19b capable of providing only two N-coordinations to each copper site. Owing to the differing stereochemical preferences20 of CuII (square pyramidal, trigonal bipyramidal, or intermediate between these two geometries) relative to CuI (tetrahedral or pyramidal) ready interconversions of these two oxidation states is expected to be facilitated by use of flexible ligands which can adjust their coordination geometry to the differing demands of the two oxidation states. To gain more insight into the reactivity of the binuclear Cu(I) complexes with dioxygen and in the dependency of the arene hydroxylation on ligand topology, we have undertaken the present investigation. In this paper we amplify our previous report19a by providing a detailed account of the synthesis and characterization of dicopper(I) complexes with L and L-NO2 and their reactivity properties with molecular oxygen. The ligand L-NO2 was synthesized to explore the possibility of stabilizing11 a peroxo-dicopper(II) intermediate. We have also explored the effect of ligand chelate ring size L′′ (Chart 1) on the reactivity of its dicopper(I) complex with O2. Absorption spectroscopic and magnetic properties of the final dicopper(II) complexes have been investigated. Experimental Section Materials and Reagents. All reagents and solvents were obtained from commercial sources and used as received unless noted otherwise. Acetonitrile (CH3CN) was dried by distillation over CaH2. Further (10) (a) Karlin, K. D.; Hayes, J. C.; Gultneh, Y.; Cruse, R. W.; McKown, J. W.; Hutchinson, J. P.; Zubieta, J. J. Am. Chem. Soc. 1984, 106, 2121. (b) Karlin, K. D.; Dahlstrom, P. L.; Cozzette, S. N.; Scensny, P. M.; Zubieta, J. J. Chem. Soc., Chem. Commun. 1981, 881. (11) Karlin, K. D.; Nasir, M. S.; Cohen, B. I.; Cruse, R. W.; Kaderli, S.; Zuberbu¨hler, A. D. J. Am. Chem. Soc. 1994, 116, 1324. (12) Mahapatra, S.; Kaderli, S.; Llobet, A.; Neuhold, Y.-M.; Palanche´, T.; Halfen, J. A.; Young, V. G., Jr.; Kaden, T. A.; Que, L., Jr.; Zuberbu¨hler, A. D.; Tolman, W. B. Inorg. Chem. 1997, 36, 6343. (13) (a) Casella, L.; Monzani, E.; Gullotti, M.; Cavagnino, D.; Cerina, G.; Santagostini, L.; Ugo, R. Inorg. Chem. 1996, 35, 7516. (b) Casella, L.; Gullitti, M.; Radaelli, R.; Gennaro, P. D. J. Chem. Soc., Chem. Commun. 1991, 1611. (c) Casella, L.; Gullotti, M.; Pallanza, G.; Rigoni, L. J. Am. Chem. Soc. 1988, 110, 4221 and references therein. (14) (a) Re´glier, M.; Jorand, C.; Waegell, B. J. Chem. Soc., Chem. Commun. 1990, 1752. (b) Re´glier, M.; AmedeI¨, E.; Tadayoni, R.; Waegell, B. J. Chem. Soc., Chem. Commun. 1989, 447. (15) Sorrell, T. N.; Garrity, M. L. Inorg. Chem. 1991, 30, 210. (16) (a) Gelling, O. J.; Meetsma, A.; Feringa, B. L. Inorg. Chem. 1990, 29, 2816. (b) Gelling, O. J.; Feringa, B. L. J. Am. Chem. Soc. 1990, 112, 7599. (b) Gelling, O. J.; van Bolhuis, F.; Meetsma, A.; Feringa, B. L. J. Chem. Soc., Chem. Commun. 1988, 552. (17) Drew, M. G. B.; Trocha-Grimshaw, J.; McKillop, K. P. Polyhedron 1989, 8, 2513. (18) Menif, R.; Martell, A. E.; Squattrito, P. J.; Clearfield, A. Inorg. Chem. 1990, 29, 4723 and references therein. (19) (a) Ghosh, D.; Lal, T. K.; Ghosh, S.; Mukherjee, R. N. J. Chem. Soc., Chem. Commun. 1996, 13. (b) Gupta, R.; Mukherjee, R. N. Inorg. Chim. Acta 1997, 263, 133. (20) (a) Schindler, S.; Szalda, D.; Creutz, C. Inorg. Chem. 1992, 31, 2255. (b) Oshio, H. J. Chem. Soc., Dalton Trans. 1990, 2985. (c) Karlin, K. D.; Sherman, S. E. Inorg. Chim. Acta 1991, 181, 111.

Ghosh and Mukherjee Chart 1

purification was achieved by KMnO4/Li2CO3 treatment21a followed by distillation over P4O10. Chloroform and dichloromethane were made acid free by washing first with sodium bicarbonate solution and then thoroughly with water, followed by storing over anhydrous CaCl2 for 24 h and distillation. A final distillation was done from calcium hydride. Tetrahydrofuran (THF) was distilled from calcium hydride. Ethanol and methanol were distilled from Mg(OC2H5)2 and Mg(OCH3)2, respectively. Diethyl ether was dried first with anhydrous CaCl2 and then refluxed with and distilled over sodium. Ethyl acetate was purified by refluxing over anhydrous K2CO3, followed by distillation. Tetra-nbutylammonium perchlorate (TBAP) was prepared/purified as before.21 1,3-Bis(bromomethyl)benzene and 1,3-bis(bromomethyl)-5-nitrobenzene were synthesized by following literature procedures.22 All airsensitive reactions were performed either in a Vacuum Atmospheres inert-atmosphere glovebox under a N2 atmosphere (Department of Chemistry, University of Minnesota) or by using standard Schlenk and vacuum line techniques. Isolation and handling of dicopper(I) complexes were carried out inside the drybox. The salts [Cu(CH3CN)4]X (X ) ClO4- or SbF6-) were prepared as described in the literature.23 Ligand Synthesis. r,r′-Bis[N-methyl-N-(2-pyridylethyl)amino]m-xylene (L). Method A.19a R,R′-m-Dibromoxylene (1.0 g, 3.74 mmol) and 2-(2-(methylamino)ethyl)pyridine (1.03 g, 7.6 mmol) were taken in CH3CN (80 mL) along with solid Na2CO3 (1.5 g, 14.0 mmol). This mixture was refluxed for 4 days. To the resulting solution were added distilled water (30 mL) and CH2Cl2 (80 mL). The organic layer was separated, dried over anhydrous Na2SO4, and kept overnight. Removal of solvent under reduced pressure afforded a brown oil. Yield: 1.3 g (92%). 1H NMR (60 MHz; CDCl3): δ 2.3 (6H, s, NMe), 2.93 (8H, s, NCH2CH2C), 3.56 (4H, s, PhCH2N), 7.0-7.9 (10H, m, aromatic protons), 8.66-8.86 (2H, d, pyridine ring protons). Some minor peaks closely associated with the peaks for the methylene and methyl protons due to some other product(s) were also observed. All our ligand preparations gave similar results. Mass spectrum [m/z (relative intensity, assignment): 374 (7.4, M+; corresponding to L), 240 (100) (the peak due to L′ was not observed due to instrument limitation)]. Positive-ion FAB mass spectrum (Figure S1, Supporting Information) [m/z (relative intensity, assignment): 375 {51, (M + H)+; corresponding to HL+}, (21) (a) Gupta, N.; Mukerjee, S.; Mahapatra, S.; Ray, R.; Mukherjee, R. N. Inorg. Chem. 1992, 31, 139. (b) Ray, M.; Mukerjee, S.; Mukherjee, R. N. J. Chem. Soc., Dalton Trans. 1990, 3635. (22) (a) Wenner, W. J. Org. Chem. 1952, 17, 523. (b) Kazuhiko, S.; Lia, R. J.; Masataka, K.; Masaki, H. J. Am. Chem. Soc. 1986, 108, 1916. (c) Sherrod, S. A.; de Costa, R. L.; Barnes, R. A.; Boekelheide, V. J. Am. Chem. Soc. 1974, 96, 1565. (23) Kubas, G. Inorg. Synth. 1979, 19, 90; 1990, 28, 68.

Modeling Tyrosinase Monooxygenase Activity 508 {9, (M + H)+; corresponding to HL′+}, 149 (100)]. On the basis of 1H NMR spectral integration the % purity of L has been determined to be ∼90%. Positive-ion FAB mass peak intensities of L and L′ closely match with the 1H NMR spectral result. Two well-separated peaks (a major and a minor) were detected in HPLC, and product ratios were determined to be closely similar to that obtained from 1H NMR and positive-ion FAB mass spectra. Method B. This methodology is adapted from the reported synthesis of a similar ligand by Gultneh et al.24 R,R′-m-Dibromoxylene (0.5 g, 1.89 mmol) was dissolved in THF (10 mL), and to it a mixture of 2-(2-(methylamino)ethyl)pyridine (0.52 g, 3.82 mmol) and Et3N (0.575 g, 5.69 mmol) in THF (10 mL) was added gradually with stirring. After 24 h of magnetic stirring at 298 K it was filtered and the light yellow colored liquid thus obtained was rotavaped to obtain a light brown oil (yield: 0.65 g, 92%). 1H NMR (60 MHz; CDCl3): δ 2.26 (6H, s, NMe), 2.9 (8H, s, NCH2CH2C), 3.56 (4H, s, PhCH2N), 6.9-7.87 (10H, m, aromatic protons), 8.4-8.6 (2H, d, pyridine ring protons). The 1H NMR spectrum (Figure S2, Supporting Information), mass spectrum [m/z (relative intensity, assignment): 374 (5.65, M+), 240 (100)], and only one peak in HPLC confirmed its purity. p-Nitro Derivative of L, L-NO2. The ligand L-NO2 was prepared by the same procedure as that followed for the synthesis of L (method B). A mixture of 2-(2-(methylamino)ethyl)pyridine (0.088 g, 0.647 mmol) and Et3N (0.098 g, 0.97 mmol) in THF (2 mL) was added gradually with stirring to a solution of 1,3-bis(bromomethyl)-5nitrobenzene (0.1 g, 0.324 mmol) in THF (2 mL). The resulting mixture was stirred at room temperature for 24 h. After filtration, the light yellow liquid thus obtained was rotavaped to obtain a yellow oil. Yield: 0.066 g (49%). 1H NMR (60 MHz; CDCl3): δ 2.23 (6H, s, NMe), 2.9 (8H, s, NCH2CH2C), 3.53 (4H, s, PhCH2N), 6.90-7.97 (9H, m, aromatic protons), 8.37-8.57 (2H, d, pyridine ring protons). r,r′-Bis[(N-methyl-N-(2-pyridylmethyl)amino]-m-xylene (L′′). mXylylenediamine (0.252 g, 1.853 mmol) and 2-pyridinecarboxaldehyde (0.397 g, 3.71 mmol) were dissolved in C2H5OH (30 mL) and refluxed for 1 h. Solvent was then removed under reduced pressure, and the oil thus obtained was extracted with CHCl3. Removal of solvent afforded the Schiff-base product as a semisolid. Yield: 0.57 g, ∼98%. 1H NMR (60 MHz; CDCl3): δ 4.80 (4H, s, PhCH2N), 7.10-8.20 (10H, m, aromatic protons), 8.36-8.70 (4H, m, pyridine ring protons and -Nd C-H protons). The Schiff base (0.60 g, 1.92 mmol) was then dissolved in CH3OH (15 mL), and to it ∼6 equiv of NaBH4 (0.44 g, 11.58 mmol) was added in portions. After each addition the reaction mixture was heated on a steam bath and subsequently cooled. This process was continued until there was no further effervescence. To the resulting mixture saturated brine solution (15 mL) was added, and this extracted 3-4 times with diethyl ether. The organic layer was collected and kept with anhydrous K2CO3, and finally solvent was removed to obtain a deep yellow oil. Yield: 0.58 g, ∼95%. 1H NMR (60 MHz; CDCl3): δ 2.20 (2H, s, -NH), 3.67-3.87 (8H, d, PhCH2N and NCH2C), 7.007.67 (10H, m, aromatic protons), 8.40-8.60 (2H, d, pyridine ring protons). The amine thus obtained (0.58 g, 1.824 mmol) was mixed with formaldehyde (2.0-2.5 mL) and formic acid (2.5-3.0 mL), and the resulting mixture was refluxed at 90 °C for 24 h. It was then cooled in an ice bath, and a saturated solution of NaOH was added to it with stirring until the pH of the solution was 12. To the resulting mixture, CHCl3 was added, and the organic layer was collected and dried over anhydrous Na2SO4. Finally the solvent was removed under reduced pressure to obtain the desired ligand L′′ as a deep brown oil. Yield: 0.545 g, ∼ 86%. 1H NMR (60 MHz; CDCl3): δ 2.10 (6H, s, -NMe), 3.40-3.60 (8H, d, PhCH2N and NCH2C), 6.83-7.73 (10H, m, aromatic protons), 8.33-8.50 (2H, d, pyridine ring protons). Mass spectrum [m/z (relative intensity, assignment): 346 (11.7, M+), 255 (100)]. r,r′-Bis[N-methyl-N-(2-pyridylethyl)amino]-4-methylphenol (L′OH).25 A solution of 2,6-bis(chloromethyl)-4-methylphenol (1.48 g, (24) Gultneh, Y.; Ahvaji, B.; Raja Khan, A.; Butcher, R. J.; Tuchagues, J. P. Inorg. Chem. 1995, 34, 3633. (25) (a) Borovik, A. S.; Papaefthymiou, V.; Taylor, L. F.; Anderson, O. P.; Que, L. Jr. J. Am. Chem. Soc. 1989, 111, 6183. (b) Karlin, K. D.; Farooq, A.; Gultneh, Y.; Hayes, J. C.; Zubieta, J. Inorg. Chim. Acta 1988, 153, 73.

Inorganic Chemistry, Vol. 37, No. 26, 1998 6599 7.2 mmol)25a in ethyl acetate (20 mL) at 273 K was added to a solution of 2-[2-(methylamino)ethyl]pyridine (1.97 g, 14.4 mmol) and Et3N (1.42 g, 14.4 mmol). This mixture was then stirred for 5 days at 298 K. After filtration, the solvent was removed and the residue was extracted with ethyl acetate (3 × 15 mL). Finally, the solvent was removed to obtain a deep brown oil. Yield: 2.75 g, ∼93%. 1H NMR (60 MHz; CDCl3): δ 2.10 (3H, s, ArMe), 2.23 (6H, s, NMe) 2.85 (8H, s, NCH2CH2), 3.56 (4H, s, PhCH2N), 6.53-7.51 (8H, m, aromatic protons), 8.38 (2H, d, pyridine ring protons), ∼10.5 (1H, br, phenolic OH). Syntheses of Complexes. Reaction of L (Method A) with Copper(I) and Subsequent Oxygenation. In dinitrogen-flushed CH2Cl2 (30 mL), solid [Cu(CH3CN)4][ClO4] (0.7 g, 2.14 mmol) was added and kept under magnetic stirring. The ligand L (0.402 g, 1.075 mmol) was dissolved in CH2Cl2 (30 mL) under a dinitrogen atmosphere and then added to the metal salt solution by a cannula. The mixture resulted in a yellow suspension of an extremely air-sensitive copper(I) complex. After stirring of the mixture for 3-4 h, it was exposed to dioxygen (1 atm; 298 K) and stirred for a further 12 h. A blue-gray compound precipitated along with a green solution. The blue-gray solid was collected by filtration, washed 3-4 times with CH2Cl2, and vacuum-dried. Yield: 0.45 g (47%, based on the composition [CuII2L(OH)(H2O)][ClO4]3‚2H2O‚0.5HCl, 3a). Solvent was removed from the green filtrate, and a CH3CN solution of the solid thus obtained was left for slow evaporation. This afforded at first only a few blue crystals ([CuII2L′(OH)2][ClO4]2, 2b) and a bulk of microcrystalline green compound. Yield: 0.32 g (40%, based on the composition [CuII2(LO)(OH)][ClO4]2, 2a). Characterization of Dicopper(II) Complexes. Green Crystals. Anal. Calcd for [CuII2(L-O)(OH)][ClO4]2 (2a), C24H30N4Cl2O10Cu2: C, 39.35; H, 4.13; N, 7.65. Found: C, 39.57; H, 4.20; N, 7.82. IR (KBr, cm-1, selected peaks): 3450 (m, ν(OH)), 1320 (s, ν(OPh)), 1100 and 620 (s, ν(ClO4-)).Conductivity (CH3CN, 10-3 M solution at 298 K): ΛM ) 280 Ω-1 cm2 mol-1. Absorption spectrum [λmax, nm (, M-1 cm-1)]: in CH3CN, 620 (170), 352 (2220), 261 (7200), 241 (7400). The first two absorptions were recorded at a concentration of 0.68 mM, and the two highest energy peaks were recorded at a concentration of 0.27 mM. When the concentration was 3 mM the peak at 352 nm shifted to 392 nm ( ) 940 M-1 cm-1). Blue Crystals. Anal. Calcd for [CuII2L′(OH)2][ClO4]2 (2b), C33H43N5Cl2O10Cu2: C, 45.68; H, 4.99; N, 8.07. Found: C, 45.52; H, 4.80; N, 7.88. IR (KBr, cm-1, selected peaks): 3600 (s, ν(OH)), 1100 and 620 (s, ν(ClO4-)). Conductivity (CH3CN, 10-3 M solution at 298 K): ΛM ) 238 Ω-1 cm2 mol-1. Absorption spectrum [λmax, nm (, M-1 cm-1)]: in CH3CN, 589 (70), 361 (985), 264 (6240). The first two absorptions were recorded at a concentration of 2.51 mM, and the highest energy peak was at a concentration of 0.53 mM. Blue-Gray Solid. Anal. Calcd for [CuII2L(OH)(H2O)][ClO4]3‚2H2O‚ 0.5HCl (3a), C24H37.5N4Cl3.5O16Cu2: C, 32.40; H, 4.22; N, 6.30. Found: C, 32.48; H, 4.15; N, 5.96. IR (KBr, cm-1, selected peaks): 3530 (m, ν(OH)), 1100 and 620 (s, ν(ClO4-)). Conductivity (CH3CN, 10-3 M solution at 298 K): ΛM ) 285 Ω-1 cm2 mol-1. Absorption spectrum [λmax, nm (, M-1 cm-1)]: 1.01 mM in CH3CN, 620 (120), 355 sh (895), 260 (5390). Organic Product Identification. To check whether hydroxylation at the ligand site has taken place, copper was removed from the bluegray solid as well as from the green compound by ammonia treatment.10a A detailed procedure for 2a is described below. The organic extracts thus obtained were identified by 1H NMR and/or positive-ion FAB mass spectral studies. A 100 mg amount of green compound 2a was dissolved in CH2Cl2 (40 mL), and to it liquor ammonia was added dropwise till the deep blue aqueous layer separated out. The organic layer was collected, washed 3-4 times with water, and finally dried over Na2SO4, and the solvent was removed under reduced pressure to get an oily yellowbrown product. The organic extract from the green compound showed the following characteristics in its 1H NMR spectrum (60 MHz, CDCl3): 2.23 (6H, s, NMe), 2.87 (8H, s, NCH2CH2C), 3.60 (4H, s, PhCH2N), 6.83-7.67 (9H, m, aromatic protons), and 8.50-8.30 (2H, d, pyridine ring protons). Identification was confirmed by its positiveion FAB mass spectrum (Figure S3, Supporting Information) [m/z

6600 Inorganic Chemistry, Vol. 37, No. 26, 1998 (relative intensity, assignment): 391 {50, (M + H)+; corresponding to H(L-OH)+}, 149 (100)]. A violet coloration was obtained when freshly prepared FeCl3 solution was added to an ethanolic solution of the organic extract obtained from the green solid 2a, confirming the presence of the phenolic-OH group in the organic product. Additionally, the 1H NMR spectrum of L′-OH (L-OH with a methyl group at 4-position of the aromatic ring) is closely similar to that obtained for L-OH. Similar ammonia treatment on the blue-gray solid 3a gave only unchanged ligand L. Isolation of Copper(I) Complexes with L (Synthesized by Method B). [Cu2L(CH3CN)2]X2 (X ) ClO4-, 1a, or SbF6-, 1b). To a stirred solution of L (0.10 g, 0.267 mmol) in CH2Cl2 (2 mL) was added solid [Cu(CH3CN)4]X (X ) ClO4- or SbF6-) (0.53 mmol). After being stirred for 10 min, the starting suspension slowly dissolved to generate a homogeneous yellow solution, at which time diethyl ether (∼5 mL) was added with vigorous stirring. This procedure resulted in the separation of a yellow solid, which was washed several times with diethyl ether, and dried under vacuum to yield a light yellow powder (for ClO4- salt, 0.21 g, 91%; for SbF6- salt, 0.27 g, 90%). 1H NMR of 1b (CD3CN, 300 MHz): δ 2.40 (6H, s, NMe), 2.75 (4H, s, NCH2CH2), 2.99 (4H, s, NCH2CH2), 3.84 (4H, s, PhCH2), 7.33 (7H, s, aromatic protons), 7.39 (1H, s, aromatic proton), 7.79 (2H, t, J ) 7.5 Hz, aromatic protons), and 8.43 (2H, s, pyridine protons). 1H NMR of 1b (CD2Cl2, 500 MHz): δ 2.27 (6H, s, CH3 of coordinated CH3CN), 2.68 (6H, s, NMe), 2.90 (4H, s, NCH2CH2), 3.02 (4H, s, NCH2CH2), 3.85 (4H, s, PhCH2), 7.33-7.48 (m, 7H aromatic protons), 7.64 (1H, s, aromatic proton), 7.82 (2H, t, J ) 8.0 Hz, aromatic protons), and 8.33 (2H, s, pyridine protons). FT IR (KBr, cm-1): for SbF6- salt, 2280 (CtN), 658 (SbF6-); for ClO4- salt, 2281 (CtN), 1093 (ClO4-). [Cu2(L-NO2)(CH3CN)2][SbF6]2 (1c). The synthesis of this compound was carried out following a similar methodology as described above. Yield: ∼ 90%. 1H NMR (CD2Cl2, 300 MHz): δ 2.26 (6H, s, NMe), 2.69 (6H, s, CH3 of coordinated CH3CN), 2.94 (4H, s, NCH2CH2), 3.05 (4H, s, NCH2CH2), 3.98 (4H, s, PhCH2), 7.32-7.36 (m, 4H aromatic protons), 7.84 (2H, s, aromatic protons), 8.01 (1H, t, J ) 8.0 Hz, aromatic proton), and 8.33 (2H, s, pyridine protons). Reaction of Pure L with Copper(I) and Subsequent Oxygenation. The reactions were carried out in four different solvents (CH2Cl2, CH3OH, CH3CN, and DMF) by using the same amount of ligand and copper(I) salt [Cu(CH3CN)4][ClO4] and by following a similar procedure. A representative case is described below. In CH2Cl2. To dinitrogen-flushed CH2Cl2 (8 mL) solid [Cu(CH3CN)4][ClO4] (0.175 g, 0.536 mmol) was added and kept under magnetic stirring. To it the ligand L (0.100 g, 0.267 mmol) dissolved in deoxygenated CH2Cl2 (8 mL) was added dropwise by a cannula. The resulting yellow suspension of an extremely air-sensitive dicopper(I) complex was stirred for 3-4 h under a dinitrogen atmosphere and finally exposed to dioxygen (1 atm; 298 K) and allowed to stir. After ∼12 h, a blue-gray solid precipitated out from a green solution. The blue-gray solid 3a was collected by filtration, washed thoroughly with CH2Cl2, and finally vacuum-dried. Yield: 0.12 g (50%). Solvent was removed from the green filtrate, and the solid thus obtained was dissolved in CH3CN and left for slow evaporation. This yielded the bulk of the microcrystalline green compound 2a. Yield: 0.06 g (30%). The yields of green solid in various solvents are as follows: CH3OH, 0.150 g (75%); CH3CN, 0.175 g (90%); DMF, 0.175 g (90%). Reaction of L′′ with Copper(I) and Subsequent Oxygenation. Solid [Cu(CH3CN)4][ClO4] (0.095 g, 0.291 mmol) was dissolved in degassed CH2Cl2 (8 mL), and the Schlenk flask was kept immersed in a CH3OH-slush bath at -60 °C. To this the ligand L′′ (0.050 g, 0.145 mmol) dissolved in CH2Cl2 (8 mL) was added and stirred under a dinitrogen atmosphere. The mixture, a yellow suspension of supposedly dicopper(I) complex, was then exposed to dioxygen at -60 °C and then gradually the flask was warmed to 298 K. This procedure was adopted to avoid the formation of a “brown solution” which otherwise was taking place when the oxygenation reaction was carried out at room temperature (this solution did not afford any tractable solid). The color of the suspension changed gradually from yellow to brown to finally green. After stirring of the reaction mixture at 298 K for 4-5 h, the color of the precipitate changed to light blue with a brown supernatant. The precipitate was filtered off, washed thoroughly with CH2Cl2, and

Ghosh and Mukherjee finally vacuum-dried to isolate a light blue solid. Yield: 0.09 g (75%, based on the composition CuII2L′′(OH)(ClO4)3(H2O)2, 3b). Anal. Calcd for C22H31N4Cl3O15Cu2: C, 32.03; H, 3.79; N, 6.79. Found: C, 32.31; H, 3.86; N, 6.65. IR (KBr, cm-1, selected peaks): 3420 (m, ν(OH)), 1140, 1100, 1080, 630, and 620 (s, ν(ClO4-)). Conductivity (CH3CN, 10-3 M solution at 298 K): ΛM ) 260 Ω-1 cm2 mol-1. Absorption spectrum [λmax, nm (, M-1 cm-1)]: 0.98 mM in CH3CN, 625 (185), 290 (sh) (5070), 259 (12 500). Caution! Perchlorate salts of compounds containing organic ligands are potentially explosiVe! Measurements. Elemental analyses were obtained from either the Indian Association for the Cultivation of Science, Calcutta, India, or National Chemical Laboratory (NCL), Pune, India. IR spectra were recorded as KBr pellets with the help of a Perkin-Elmer 1320 or on a Mattson Polaris FTIR spectrophotometers. Electronic spectra were measured with a Perkin-Elmer Lambda 2 spectrophotometer, and solidstate electronic spectra (reflectance spectra) were measured using paraffin oil. Low-temperature UV-vis spectra were recorded (Department of Chemistry, University of Minnesota) on a Hewlett-Packard HP8452A diode array spectrophotometer (190-820 nm scan range) by using a custom manufactured vacuum dewar equipped with quartz windows. Low temperatures were achieved by adding liquid nitrogen to a CH3OH bath. The bath temperature was monitored by using a resistance thermocouple probe (Fluke 51 K/J thermometer). 1H NMR spectra were obtained on a PMX-60 JEOL (60 MHz) instrument or Bru¨ker WP-80 (80 MHz) or VXR-500 (500 MHz) NMR spectrometers using CDCl3, CD2Cl2, or CD3CN solutions. Chemical shifts (in ppm) are referenced to the residual solvent peak(s) or TMS. HPLC experiments were performed on a Pharmacia LKB model 2248, using a reverse-phase C18 column at a flow rate of 1 mL/min. Elution was achieved by 40:60 (v/v) CH3CN/CH3OH, and its profile was monitored by Pharmacia LKB model 2141 at 367 nm. Solution electrical conductivity measurements were made with an Elico (Hyderabad, India) Type CM 82 T conductivity bridge. Solution magnetic susceptibility measurements were made by the usual NMR method26 with a PMX60 JEOL (60 MHz) spectrometer and made use of the paramagnetic shift of the methyl protons of CH3CN or TMS reference as the measured NMR parameter. Diamagnetic corrections27 were taken from literature tabulations. Mass spectra were obtained with a Finnigan MAT-1020-B 70-eV mass spectrometer at the NCL, Pune, India. Positive-ion fast atom bombardment (FAB) mass spectra were recorded at the Central Drug Research Institute, Lucknow, India, on a JEOL SX 102/DA-6000 mass spectrometer/data system using argon gas ionization in a matrix of m-nitrobenzyl alcohol as the matrix and CH3CN as solvent. Variable-temperature magnetic susceptibility measurements were obtained on polycrystalline samples (powdered form) by the Faraday method using a locally built susceptometer. The system consists of an electromagnet (Polytronic Corporation, Mumbai, India), a microbalance (Sartorius, Go¨ttingen, Germany), and a closed-cycle cryostat (CTI cryogenics). The calibration of the system and the details of measurements are already reported in the literature.28 Effective magnetic moments were calculated from µeff ) 2.828[χMT]1/2, where χM is the corrected molar susceptibility. The diamagnetic contributions were -368 × 10-6 cm3 mol-1 for 2a, -473 × 10-6 cm3 mol-1 for 2b, -445 × 10-6 cm3 mol-1 for 3a, and -402 × 10-6 cm3 mol-1 for 3b. The data were fitted to the Bleany-Bowers expression (eq 1) for two

χM ) 2Nβ2g2/3kT[1 + 1/3 exp(-2J/kT)]-1(1 - F) + Nβ2g2F/2kT + 2NR (1) interacting S ) 1/2 centers developed under the usual isotropic (Heisenberg) exchange Hamiltonian H ) -2JS1‚S2.27 Inclusion of terms for the temperature-independent paramagnetic susceptibility (TIP) and for possible sample contamination of monomeric copper(II) impurity exhibiting Curie behavior yielded the appropriate equation (eq 1). In (26) Evans, D. F. J. Chem. Soc. 1959, 2003. (27) O’Connor, C. J. Prog. Inorg. Chem. 1982, 29, 203. (28) Ray, M.; Ghosh, D.; Shirin, Z.; Mukherjee, R. N. Inorg. Chem. 1997, 36, 3568.

Modeling Tyrosinase Monooxygenase Activity

Figure 1. CD2Cl2.

Inorganic Chemistry, Vol. 37, No. 26, 1998 6601

1

H NMR spectrum of [Cu2L(CH3CN)2][SbF6]2 (1b) in

this expression, N, g, and k have their usual meaning, F is the fraction of monomeric impurity, and NR is the temperature-independent paramagnetism; 2J is the energy differnce between the singlet and triplet states, and χM is the molar susceptibilty per dimer. A nonlinear regression analysis was carried out with J, g, and F as floating parameters. X-band EPR spectra were recorded with a Varian E-109 C spectrometer fitted with a quartz dewar for measurements at liquid dinitrogen temperature. The spectra were calibrated with the help of DPPH (g ) 2.0037).

Results and Discussion Syntheses of Ligands and Dicopper(I) Complexes. The dinucleating ligands (Chart 1) were prepared by standard methods (Experimental Section). To minimize the complications in handling with an impure ligand L (method A),19a in the meantime we successfully developed a methodology (method B) for the synthesis of pure ligand L. Isolation of the dicopper(I) complexes were achieved by the addition of 2 equiv of [Cu(CH3CN)4]X (X ) ClO4- or SbF6-) to the solution of appropriate pure ligand in CH2Cl2. The composition/purity of the dicopper(I) complexes were judged by 1H NMR spectroscopy for [Cu2L(CH3CN)2]X2 (X ) ClO4- (1a), SbF6- (1b)) and [Cu2(L-NO2)(CH3CN)2][SbF6]2 (1c). Additional proof of their compositions were obtained from FT IR (1a,b). The 1H NMR spectra of 1b,c are displayed in Figure 1 and Figure S4, Supporting Information, respectively. Reactivity of [Cu2L(CH3CN)2]X2 (X ) ClO4- (1a), SbF6(1b)) and [Cu2(L-NO2)(CH3CN)2][SbF6]2 (1c) with Dioxygen at -80 °C. It has been proposed1,3,5,6,9-18 that for regiospecific aromatic ring hydroxylation by copper with m-xylyl-based ligand systems, the reaction proceeds through the formation of a (µη2:η2-peroxo)dicopper(II) intermediate which has an intense purple/orange-red color depending on the nature of the ligand system. This peroxo species is unstable at higher temperatures but fairly stable for most of the systems at low temperatures, ∼-80 °C.11,12 Kitajima et al.29 determined the structure of this novel dicopper-dioxygen adduct with their tris(3,5-diisopropylpyrazolyl)borate ligand. To investigate possible formation of the peroxo species, with the present ligand L, CH2Cl2 and THF solutions of the dicopper(I) complexes were exposed to molecular oxygen at -80 °C and absorption spectral behavior was investigated at this temperature. In both solvents the solution turned green within the time of exposure and an absorption band appeared at 352 nm ( ∼ 2000 M-1 cm-1), which did not change on (29) Kitajima, N.; Fujisawa, K.; Fujimoto, C.; Moro-oka, Y.; Hashimoto, S.; Kitagawa, T.; Toriumi, K.; Tatsumi, K.; Nakamura, A. J. Am. Chem. Soc. 1992, 114, 1277 and references therein.

Figure 2. Oxygenation reaction of [Cu2L(CH3CN)2][SbF6]2 (1b) in CH2Cl2 at - 80 oC. The lower trace (solid line) is for the dicopper(I) compound.

warming the solution to room temperature (Figure 2). It is thus clear that the peak at 352 nm is not due to peroxo f Cu(II) charge-transfer11,12 but due to PhO-/OH- f Cu(II) chargetransfer transition, originating due to the formation of the phenoxo- and hydroxo-bridged dicopper(II) complex (vide infra). As here the oxygenation reactions are instantaneous, we have not been able to determine the reaction stoichiometry of CuI2/O2 at room temperature from the measurements of the uptake of dioxygen (manometry). The dicopper(I) complexes 1a,b grossly resemble Karlin’s system with the primary difference that Karlin’s ligand provided two ethylpyridine arms from each aliphatic nitrogen and only one such arm is present in our ligand. Using a p-nitro derivative of the parent ligand, Karlin et al.11 spectroscopically identified the peroxo species which was fairly stable at -80 °C. The presence of an electron-withdrawing -NO2 substituent in the para-position made the xylyl ring electron poor and hence decreased the rate of subsequent electrophilic attack of the peroxo species to the aromatic ring. Hoping along the same line, we synthesized the p-nitro derivative of L, L-NO2, and its dicopper(I) complex. However, when this complex 1c dissolved in either CH2Cl2 or THF was exposed to O2 at -80 °C, we could not detect any peroxo intermediate. The otherwise kinetically favorable hydroxylation of the aromatic ring was observed. In essence, it implies that the peroxo intermediate is of insufficient stability to be observed because of the low barrier for its subsequent reaction. Both Karlin’s complexes and these ones oxygenate quickly at low temperature. To extract information regarding relative stabilization of CuII over CuI oxidation state (electronic effect), in a given solvent, we investigated briefly the redox behavior of the dicopper(I) complex of L and compared it with that of Karlin’s system. When examined by cyclic voltammetry under anaerobic conditions, complex 1a (generated in DMF) exhibits a single oxidative redox process with peak-to-peak separation of 240 mV, for the redox couple [CuII2L(CH3CN)2]4+/ [CuI2L(CH3CN)2]2+ at E1/2 ) - 0.26 V vs SCE. A typical CV scan is displayed in Figure 3. The E1/2 value for the Karlin’s system is reported to be 0.31 V vs Ag/AgCl,30 i.e., 0.29 V vs

6602 Inorganic Chemistry, Vol. 37, No. 26, 1998

Figure 3. Cyclic voltammogram of solution-generated [Cu2L(DMF)2][ClO4]2 in DMF at a platinum working electrode (V ) 50 mV s-1).

SCE. Most interestingly, the E1/2 value for our system is 550 mV more cathodic than that of Karlin’s system. The CV result convincingly justifies the observed enhanced oxygen sensitivity of our dicopper(I) complexes. It should be emphasized here that although the reduction potential is a good predictor of the reactivity of the copper(I) complex toward dioxygen, it is not directly correlated with the feasibility for the formation of the dioxygen adduct, since the redox potentials are defined for outersphere-type electron-transfer reactions.1a Reaction between L (Prepared by Method A) and [Cu(CH3CN)4][ClO4] and Subsequent Oxygenation in CH2Cl2 at Room Temperature. In the initial stages of this work, synthesis of L was achieved by method A. From 1H NMR and positive-ion FAB mass spectra it was revealed that it contains some minor impurity. This impurity was eventually identified19a from the X-ray structure of the isolated dicopper(II) complex (2b). Initial synthetic reactions of L with [Cu(CH3CN)4][ClO4] [0.4 g of L and 0.7 g of the copper(I) salt (Cu:L ) 2:1)] were carried out in CH2Cl2 (∼30 mL) under a dinitrogen atmosphere. The yellow solution with a yellow suspension19a thus obtained was exposed to dioxygen. After 12 h, the reaction mixture turned dark green and afforded green phenoxo- and hydroxo-bridged dicopper(II) complex (2a) as the major isolated solution product with a very small amount of light blue dihydroxo-bridged dicopper(II) complex 2b. Interestingly, an appreciable amount of CH2Cl2-insoluble blue-gray compound, supposedly irreversible oxidation product, was also isolated.19a Reactivity of Solution-Generated [Cu2L(CH3CN)2][ClO4]2 (1a) with Dioxygen at Room Temperature. (a) In CH2Cl2. Following a similar reaction sequence as stated in the previous section, we isolated only two dicopper(II) compounds: green microcrystalline (2a) and blue-gray (3a).31 (b) In CH3CN, DMF, and CH3OH. In these solvents the reactions were carried out under identical conditions as that in CH2Cl2. In these cases, on exposure to dioxygen, initially formed yellow solution gradually changed to dark green, which eventually allowed the isolation of the green microcrystalline solid 2a. Interestingly, in these solvents no blue-gray solid was formed. (c) Effect of Solvent on the Aromatic Ring Hydroxylation. The role of solvent is expected to be of significance, since the availability of the open coordination site is depended on the coordinating ability of the solvents.1a Moreover, the stability of the dioxygen adduct is expected to be influenced by the interaction between the dioxygen adduct and the solvent. (30) Karlin, K. D.; Tyekla´r, Z.; Farooq, A.; Haka, M. S.; Ghosh, P.; Cruse, R. W.; Gultneh, Y.; Hayes, J. C.; Toscano, P. J.; Zubieta, J. Inorg. Chem. 1992, 31, 1436. (31) Reactions between isolated dicopper(I) complexes and dioxygen give rise to only phenoxo-/hydroxo-bridged product.

Ghosh and Mukherjee However, it is to be noted that the actual coordination number at Cu(I) centers in the present investigation is unsure, given the solvent coordination from Cu(CH3CN)4+. From the results presented in the previous section, we infer that the extent of aromatic ring hydroxylation with our binucleating ligand L is very much dependent on the nature of the medium in which the oxygenation reaction has been performed. The formation of the phenoxo- and hydroxo-bridged dicopper(II) complex 2a, which is the aromatic ring hydroxylation product, varies with the polarity of the solvent. In polar solvents32 like CH3CN (dielectric constant,  ) 37.5) and DMF ( ) 36.7), the extent of hydroxylation was almost quantitative, which decreased in a comparatively less polar CH3OH ( ) 32.6) and was considerably less in a nonpolar solvent like CH2Cl2 ( ) 9.1). Reaction between L′′ and [Cu(CH3CN)4][ClO4] and Subsequent Oxygenation in CH2Cl2. The ligand L′′ was synthesized to specifically pinpoint the effect of ligand topology, to bring about aromatic ring hydroxylation with our parent m-xylylbased binucleating ligand L. It is to be noted that on coordination while L gives rise to six-membered chelate ring, L′′ provides five-membered chelate ring. Treatment of L′′ with 2 equiv of [Cu(CH3CN)4][ClO4] in CH2Cl2 under dinitrogen at -60 °C, followed by exposure to dioxygen and slowly warming up the reaction mixture to 298 K, resulted in an immediate color change of the suspension from yellow to light blue. We confirmed that hydroxylation of the benzene ring of the ligand had not occurred, by isolating the ligand from the isolated dicopper(II) complex 3b (vide infra). The organic extract has the same 1H NMR spectral feature as that in L′′. Identification of the Copper(II) Products: Green 2a, Blue 2b, Blue-Gray 3a, and Light Blue 3b. IR spectra of 2a,b display absorptions due to ν(O-H) and ν(ClO4-). The complex 2a displays an additional phenolate ν(C-O) absorption.19b Elemental analyses, IR, and solution electrical conductivity data33 of the dicopper(II) complexes are in agreement with the following formulations: [Cu2(L-O)(OH)][ClO4]2, 2a; [Cu2L′(OH)2][ClO4]2, 2b. Complex 3a, which is an “irreversibly oxidized product”,11,12,34-38 displays IR absorption due to water and/or hydroxo coordination.39 Interestingly, for 3b split absorptions due to bridging ClO4- coordination are clearly observable.40 On the basis of elemental analysis, solution electrical conductivity data (1:2 electrolyte),41 and additional solution behavior,42 we propose the unhydroxylated product 3a to have the composition [Cu2L(OH)(H2O)][ClO4]3‚2H2O‚0.5HCl. Final (32) Sawyer, D. T.; Roberts, J. L., Jr. Experimental Electrochemistry for Chemists; Wiley: New York, 1974; pp 204-207. (33) Geary, W. J. Coord. Chem. ReV. 1971, 7, 81. (34) Nelson, S. M.; Esho, F.; Lavery, A.; Drew, M. G. B. J. Am. Chem. Soc. 1983, 105, 5693. (35) Sorrell, T. N.; Borovik, A. S. J. Chem. Soc., Chem. Commun. 1984, 1489. (36) (a) Nasir, M. S.; Cohen, B. I.; Karlin, K. D. J. Am. Chem. Soc. 1992, 114, 2482. (b) Sanyal, I.; Tahir-Mahroof, M.; Nasir, M. S.; Ghosh, P.; Cohen, B. I.; Gultneh, Y.; Cruse, R. W.; Farooq, A.; Karlin, K. D.; Liu, S.; Zubieta, J. Inorg. Chem. 1992, 31, 4322. (c) Karlin, K. D.; Cruse, R. W.; Gultneh, Y.; Farooq, A.; Hayes, J. C.; Zubieta, J. J. Am. Chem. Soc. 1987, 109, 2668. (d) Karlin, K. D.; Gultneh, Y.; Hayes, J. C.; Zubieta, J. Inorg. Chem. 1984, 23, 519. (37) Davies, G.; El-Sayed, M. A.; Henary, M. Inorg. Chem. 1987, 26, 3266. (38) Casella, L.; Carugo, O.; Gullotti, M.; Garofani, S.; Zanello, P. Inorg. Chem. 1993, 32, 2056. (39) Drew, M. G. B.; Nelson, J.; Esho, F.; McKee, V.; Nelson, S. M. J. Chem. Soc., Dalton Trans. 1982, 1837. (40) Rosenthal, M. R. J. Chem. Educ. 1973, 50, 331. (41) Complex 3a is expected to behave as 1:3 electrolyte, not the observed 1:2 electrolytic behavior.33 It is proposed to be due to their rearrangements in CH3CN solution: 3a to the doubly hydroxy-bridged complex, [Cu2L(OH)2][ClO4]2‚2H2O.

Modeling Tyrosinase Monooxygenase Activity

Inorganic Chemistry, Vol. 37, No. 26, 1998 6603

Figure 4. Schematic representations of the structures of the dicationic parts of [Cu2(L-O)(OH)][ClO4]2 (2a) and [Cu2L′(OH)2][ClO4]2 (2b). Table 1. X-ray Structural Metric Parameters19a and 2J Values for 2a,b Cu‚‚‚Cu (Å) torsion angle of the Cu2O2 unit (deg) average angles between the planes N-Cu-N and O-Cu-O (deg) 2J (cm-1)

2a

2b

2.999 (1) ∼7 ∼6

3.004 (2) ∼12 ∼2

-440

-258

proof of the presence of a hydroxo bridge and an aquo bridge came from its temperature-dependent magnetic susceptibility measurements and spectral titration experiments (vide infra). It is worth mentioning here that Karlin et al.11 proposed a single hydroxo-bridged tricationic species, similar to 3a, for their irreversibly oxidized product. In the positive-ion FAB mass spectra of 3a,b, intense peaks were observed at m/z values of 537 and 509, respectively, corresponding to the presence of species {H[CuII2L(OH)(H2O)]}4+/{[CuII2L(H2O)2]}4+ and {[CuII2L′′(H2O)2]4+}, respectively (Figure S5, Supporting Information). Unfortunately, all the attempts to grow crystals of 3a,b suitable for X-ray analysis and thus fully characterize these complexes in the solid state have been unsuccessful so far. Even we could not obtain elemental analysis data better than that reported here. Description of the Structures. (a) [Cu2(L-O)(OH)][ClO4]2 (2a). A schematic view of the cationic part of 2a and metric parameters pertinent to this work are in Figure 4 and Table 1, respectively. The X-ray structure of 2a19a underscores the incorporation of two O atoms into the complex: one into the aryl hydrogen bond and the other into the hydroxy bridge. The average Cu-O (phenoxo: 1.961 Å; hydroxo, 1.913 Å), Cu-N (amine, 2.017 Å; pyridine, 2.003 Å), and Cu- - -Cu distances19a are closely similar to related complexes.10,16,17 Considering the bond lengths and angles, it reveals that the geometry around each copper(II) ion in 2a is distorted square planar. The Cu atoms lie more or less in the plane containing the O and the N atoms [one Cu atom being ∼0.04 Å and the other atom being ∼0.02 Å out of plane]. The two Cu(II) ions are in almost identical coordination environments. (b) [Cu2L′(OH)2][ClO4]2 (2b). Figure 4 depicts a schematic view19a of the cationic part of 2b, and pertinent bonding parameters are given in Table 1. The trend in the Cu-N (amine, 2.045 Å; pyridine, 1.997 Å) and Cu-O (1.925 Å) bonding parameters is closely similar to that observed in 2a. The average Cu-O bond length and Cu-O-Cu angle are typical of the presence of dihydroxy bridges.44-46 Most interestingly, the (42) We were rather surprised to detect chloride ion, as the AgCl precipitate, from the hot aqueous nitric acid solution of 3a. Additionally, we could detect the presence of Cl- ion as trapped HCl from the acidic nature of suspended aqueous solution of this complex (gravimetric analysis of chloride as AgCl: calcd, 2.00; found, 1.98). Dichloromethane must be the likely source of the chloride ion. It should be mentioned here that Karlin et al.43 and Sorrell et al.15 also found that their copper(I) complexes react with CH2Cl2 resulting in the formation of chlorocopper(II) compounds.

Cu2O2 unit is bent (hinge distortion). The copper atom is almost in the coordination plane (out of plane displacement is only 0.02 Å). Visible Absorption Spectra of the Copper(II) Complexes: Green (2a), Blue (2b), Blue-Gray (3a), and Light Blue (3b). The electronic spectrum of 2a (Figure S6, Supporting Information) shows a ligand field band at 620 nm. A ∼3 mM solution of 2a gives an intense peak at 392 nm which shifts on dilution (∼0.7 mM) to 352 nm. The exact cause of this behavior is not very clear. The high-energy transition is due to PhO- f Cu(II) and/or OH- f Cu(II) ligand-to-metal charge-transfer (LMCT) origin.10-13,15-18 The absorption spectrum of a ∼2.5 mM solution of 2b (Figure S6, Supporting Information) displays a ligand field band at 589 nm and an intense peak at 361 nm, typical of an OH- f Cu(II) charge-transfer band.15,38 Intraligand charge-transfer bands were also observed for both the complexes. The bluish gray irreversible oxidation product 3a instantaneously changes in CH3CN to generate a bluish green solution, implying a structural change. This was also clear from the difference in its spectral behavior in the solid state and in solution. The solid-state reflectance spectrum (in paraffin oil) exhibits a broad band at ∼560 nm, and when the sample was diluted, only two shoulders at 365 and 277 nm were observed. In contrast, a ∼1 mM CH3CN solution displays a ligand field band at 620 nm, a weak shoulder at 355 nm, and a peak at 260 nm of intraligand origin. It is interesting to note here that the position of the band at 355 nm varies with concentration. Moreover, the shoulder is not observable in very dry CH3CN; however, on dilution this shoulder becomes prominent, probably due to attainment of additional hydroxo-bridging (vide infra). The band at ∼355 nm is supposedly due to OH- f Cu(II) charge transfer.15,38 The behavior in not so dry CH3CN is thus understandable. Interestingly, no color change is observed when 3b is dissolved in CH3CN. The spectral behavior of 3b (d-d transition at 625 nm and a shoulder of charge-transfer origin at 290 nm) suggests that each copper(II) center is four-coordinate47 with no hydroxo bridging as in 3a. To look into the nature of proposed bridging group(s) (aquo/ hydroxo) in 3a, the following controlled experiments were

performed. It has been observed that addition of 1 equiv of HClO4 (in CH3CN) the band at ∼ 355 nm disappears. Ad(43) (a) Jacobson, R. R.; Tyekla´r, Z.; Karlin, K. D. Inorg. Chim. Acta 1991, 181, 111. (b) Jacobson, R. R.; Tyekla´r, Z.; Farooq, A.; Karlin, K. D.; Liu, S.; Zubieta, J. (44) Lee, S. C.; Holm, R. H. Inorg. Chem. 1993, 32, 4745. (45) Martens, C. F.; Schenning, A. P. H. J.; Feiters, M. C.; Heck, J.; Beurskens, G.; Beurskens, P. T.; Steinwender, E.; Nolte, R. J. M. Inorg. Chem. 1993, 32, 3029. (46) (a) Ruiz, E.; Alemany, P.; Alvarez, S.; Cano, J. Inorg. Chem. 1997, 36, 3683. (b) Charlot, M. F.; Jeannin, S.; Jeannin, Y.; Kahn, O.; Lucrece-Abaul, J.; Martin-Frere, J. Inorg. Chem. 1979, 18, 1675. (c) Crawford, V. H.; Richardson, H. W.; Wasson, J. R.; Hodgson, D. J.; Hatfield, W. E. Inorg. Chem. 1976, 15, 2107. (d) Hay, P. J.; Thibeault, J. C.; Hoffmann, R. J. Am. Chem. Soc. 1975, 97, 4884. (47) Hathaway, B. J. ComprehensiVe Coordination Chemistry; Wilkinson, G., Ed.; Pergamon: New York, 1987; Vol. 5, pp 533-774.

6604 Inorganic Chemistry, Vol. 37, No. 26, 1998 ditionally, it causes a red shift of the d-d band. However, on addition of 4 equiv of an aqueous solution (40%) of TBA+OHin CH3CN the band reappears (Figure S7, Supporting Information). This demonstrates the presence of a hydroxo group in 3a, which rearranges to dihydroxo species in solution (vide infra). For 3b, we have noticed that addition of 1 equiv of the above base causes decomposition of the complex and addition of 2 equiv of acid generates a weak shoulder at ∼340 nm. With the results at hand we are not in a position to propose the structure of 3b. Magnetism of Copper(II) Complexes. (a) Solid-State Behavior. The structurally chatracterized complexes 2a,b have given us an unique opportunity to study the magnetic behavior of two dicopper(II) complexes with phenoxo-/hydroxo- and bis(hydroxo)-bridging having closely similar terminal ligation. Therefore, there is a possibility to arrive at a magneto-structural trend. The detailed temperature-dependent magnetic studies of 2a,b, and 3a,b in the solid state (Faraday method) were undertaken to elucidate the extent of magnetic exchange interactions in these systems. For 2a,b the measurements were carried out in the temperature range 25-300 K. In the case of 2a, the value of µeff/Cu at 300 K is found to be 1.13 µB which decreased sharply to 0.35 µB at 100 K and 0.24 µB at 25 K. A plot of molar susceptibilty (per dimer) versus temperature is illustrated in Figure 5. The behavior is typical of a strongly antiferromagnetically coupled system. A sharp rise in χM at low temperatures indicates the presence of small amount of paramagnetic impurity. The best fit obtained using eq 1 (Figure 5) gave 2J ) - 440 cm-1, g ) 2.05, and F ) 0.016. The large negative value of J indicates a strong coupling between the two copper centers, expected for these types of systems.48,49 In the related system of Karlin et al. even stronger coupling was observed (2J ) 600 cm-1).48 For 2b, the value of µeff/Cu at 300 K is 1.50 µB and it decreases to 0.72 µB at 100 K and finally to 0.43 µB at 25 K. This behavior is closely similar to that of 2a. Figure 5 displays the plot of molar susceptibility (per dimer) versus temperature with the following values: 2J ) - 258 cm-1; g ) 2.13; F ) 0.054. The extent of magnetic coupling is comparable to closely related systems.46 This result reveals that in 2b the extent of coupling is much reduced than that in 2a. The pronounced effect of the extent of magnetic coupling in going from 2a (phenoxo/hydroxo-bridge) to 2b (dihydroxo bridge) for a closely similar ligand environment is worth noticing. For 3a, the value of µeff/Cu is 1.86 µB at 300 K. In fact, the observed value is that expected for a mononuclear magnetically dilute copper(II) ion.47 However, temperature-dependent magnetic susceptibility measurements revealed that the µeff/Cu value at 300 K gradually decreases to 1.43 µB at 81 K. A plot of molar susceptibilty (per dimer) versus temperature along with nonlinear least-squares fitting of the data using eq 1 is displayed in Figure 6. This plot yielded the following values: 2J ) - 70 cm-1; g ) 2.15; F ) 0.030. The temperature-dependent behavior of this complex is also that expected for an antiferromagnetically coupled system. It should be noted, however, that for 3a the extent of magnetic coupling is much reduced. Comparing the magnetic behavior of 3a with that of a structurally characterized dicopper(II) complex with an aquo bridge in addition to a hydroxy in a macrocyclic environment39 gives confidence to our proposed structure. For 3b, the value of µeff/Cu is 1.98 µB (48) Karlin, K. D.; Farooq, A.; Hayes, J. C.; Cohen, B. I.; Rowe, T. M.; Sinn, E.; Zubieta, J. Inorg. Chem. 1987, 26, 1271. (49) Grzybowski, J. J.; Merrell, P. H.; Urbach, F. L. J. Inorg. Chem. 1978, 17, 3078.

Ghosh and Mukherjee

Figure 5. Plots of molar susceptibility (χM per dimer) versus temperature for polycrystalline samples of (a) [Cu2(L-O)(OH)][ClO4]2 (2a) and (b) [Cu2L′(OH)2][ClO4]2 (2b). The solid lines result from a least-squares fit of the susceptibility data to eq 1. See the text for fitting parameters.

at 300 K and is gradually decreases to 1.77 µB at 81 K. A similar analysis gives rise to the following parameters: 2J ) - 4 cm-1; g ) 2.15; F ) 0.00025. Thus the extent of magnetic coupling follows the order 2a > 2b > 3a > 3b. (b) Solution-State Behavior. The effective magnetic moments for 2a and 3a,b in CH3CN solution (300 K) were determined by using the NMR method.26 The µeff/Cu value for 2a was found to be 1.05 µB, in conformity with the solid-state value. Most interestingly, the value of µeff/Cu for 3a is 1.55 µB, which is considerably less than the solid-state value (1.86 µB). This could be due to the following reason. In CH3CN solution the water molecule bridged to copper(II) centers reorganizes to provide an additional hydroxo bridge between the two copper(II) centers, {Cu2L(OH)2}2+ (vide supra). This in turn provides a better pathway for magnetic coupling and thus results in lower value of µeff/Cu. In fact, the data decrease with time. After 24 h, the value becomes 1.41 µB. This proposition provides an explanation for its observed conductivity data (1:2 electrolytic behavior). Due to difficulty in isolating

Modeling Tyrosinase Monooxygenase Activity

Figure 6. Plots of molar susceptibility (χM per dimer) versus temperature for solid samples of [Cu2L(OH)(H2O)][ClO4]3‚2H2O‚ 0.5HCl (3a). The solid line results from a least-squares fit of the susceptibility data to eq 1. See the text for fitting parameters.

pure samples of 2b in bulk quantity the solution-state magnetic moment value for this complex was not determined. For 3b the corresponding value is 1.70 µB, which is appreciably lower than that observed in the solid state (1.98 µB). (c) Magneto-Structural Correlations. It is well documented now that the type and magnitude of magnetic exchange interactions in dinuclear complexes depend on the bridge identity, the M- - -M separation, the bond angles at the bridging atoms, the dihedral angle between the planes containing the copper(II) ions, the metal-bridging ligand bond lengths, and the metal ion stereochemistry.46 From the variable-temperature magnetic susceptibility data of 2a,b, it is clear that in both the complexes the two copper(II) centers are antiferromagnetically coupled. However, the coupling is much more pronounced in the former phenoxo/hydroxodicopper(II) complex (2J ) - 440 cm-1) than in the latter bis(µ-hydroxo)dicopper(II) complex (2J ) -258 cm-1). A closer look at Table 1 reveals that, in both the complexes, the Cu- - - Cu distance, the stereochemistry around each Cu center, and the average Cu-OR-Cu bond angle at the bridging atoms are comparable. The metal-bridging ligand bond lengths are slightly longer for the phenoxo-/hydroxo-bridged complex 2a than the dihydroxo-bridged complex 2b. The fact is that the Cu2O2 unit in 2b deviates more from planarity than that of 2a. This difference can provide a reasonable explanation to the difference in the magnetic behavior of the two complexes. It can be suggested that the lesser coupling in 2b is attributable to the fact that two copper(II) ions do not lie in one plane. When the metal ion moves into the plane, overlap between the metalbased dx2-y2 orbitals and the oxygen-based sp2 hybrid orbitals is increased. Since this σ framework represents the dominant pathway for the superexchange mechanism, the enhanced overlap should result in an increase in the antiferromagnetic interaction. EPR Spectra. To substantiate the magnetic behavior of 3a,b we investigated their EPR spectral properties. For 3a, the roomtemperature solid-state spectrum shows features typical for a tetragonal copper(II) (g|| ) 2.26, g⊥ ) 2.06, A|| ) 165 G).47 At 77 K in the solid-state as well as in CH3CN solution, the signal is comparatively weak (cf. magnetic studies). Solid samples of

Inorganic Chemistry, Vol. 37, No. 26, 1998 6605 3b at 300 K exhibit a broad signal (g|| ) 2.30, g⊥ ) 2.07, A|| ) 160 G). At 77 K both in the solid state and in CH3CN solution, the samples exhibited very weak signal with gav ) 2.06, suggesting weak coupling (cf. magnetic studies). Solution-State Structure of the Phenoxo-/Hydroxo-Bridged Dicopper(II) Complex 2a by 1H NMR Spectroscopy. To see whether the solid-state structure is retained in solution, 1H NMR spectral behavior of 2a was investigated in CD3CN. The spectral behavior (the spectrum ranges from +26 to -6 ppm) in CD3CN is shown in Figure S8, Supporting Information. It is obvious that the bridged structure of 2a is retained in solution. Summary/Overview. With the synthesis of an interesting series of m-xylyl-based ligands L (L′), L-NO2, and L′′, capable of providing only two nitrogen coordinations to each copper center, the oxygenation of the dicopper(I) complexes have been systematically investigated. Rapid decomposition to aromatic ring-hydroxylated product even at -80 °C implies that the energy barrier for conversion of intermediate dicopper(II)peroxo to such species is small. Several groups have tried to throw light on the oxygenation behavior as a function of the nature of solvent.11-13 The observed higher hydroxylation yield in more polar solvent is probably due to better solvation of polar transition states and/ or intermediates.50 The present studies pinpoint (i) the effect of ligand structure, i.e., a six-membered chelate ring-forming ligand (L) gives rise to aromatic ring hydroxylation whereas a five-membered chelate ring-forming ligand (L′′) gives only irreversible oxidation, (ii) the effect of appropriate positioning (geometry) of the xylyl ring of the ligand, i.e., compared to L, the failure of the ligand L′ to undergo hydroxylation reaction, and (iii) the effect of solvent polarity, i.e., a less polar solvent such as CH2Cl2 at 298 K gives rise to both hydroxylated as well as unhydroxylated product whereas a more polar solvent such as CH3CN or DMF gives only hydroxylated product, on the reactivity properties of present dicopper(I) complexes with molecular oxygen. Experiments to carefully look at a comparative kinetic data on i the solvent dependence and ii temperature dependence of the aromatic hydroxylation are being planned. Acknowledgment. Financial support by the Department of Science and Technology and the Council of Scientific and Industrial Research, Government of India, is gratefully acknowledged. Our sincere thanks are due to Professor W. B. Tolman for extending his laboratory facilities to carry out isolation and characterization of dicopper(I) complexes and low-temperature oxygenation studies. We are grateful to Dr. Samiran Mahapatra for performing such key experiments. We thank Rajeev Gupta for technical assistance, Pulakesh Mukherjee for some preliminary experiments with the synthesis of L′′, and Dr. Nishi Gupta for the synthesis of L′-OH. Supporting Information Available: Mass spectra (FAB) of ligand L (from method A) (Figure S1), 1H NMR spectrum of ligand L (from method B) (Figure S2), mass spectra (FAB) of L-OH (Figure S3), 1H NMR spectrum of 1c in CD2Cl2 (Figure S4), mass spectra (FAB) of 3a,b (Figure S5), UV-vis spectra of 2a,b in CH3CN (Figure S6), acid/ base titration of 3a in CH3CN (Figure S7), 1H NMR spectrum of 2a in CD3CN (Figure S8), and magnetic data for 2a,b, and 3a,b (Tables S1S4) (13 pages). Ordering information is given on any current masthead page. IC9713689 (50) The reaction resembles a simple electrophilic aromatic substitution and might be expected to proceed via a cationic intermediate akin to a “σ complex” that would be preferentially stabilized in polar solvent. We are grateful to one of the reviewers for suggesting this.